1. Field of the Invention
The present invention relates to methods for expression screening of filamentous fungal cells transformed with a DNA library.
2. Description of the Related Art
Commercial production of proteins frequently relies on use of recombinant host cells for expression. Prior to commercial production, research and development aimed at selecting and/or improving proteins of interest also involves use of such recombinant host cells. Screening projects, such as screening protein variants encoded by mutants of a specific gene, or screening libraries of genes from a genomic or cDNA library, are often restricted to hosts outside filamentous fungi. The most frequently utilized hosts are yeasts and bacteria, which are well-suited for high-throughput methods required for screening. Typically, thousands to hundreds of thousands of polynucleotide fragments are transformed into hosts, these transformants are cultured in a medium, and the cultures are screened for identification of proteins of interest. Often the goal is to identify or engineer a protein with improved properties, e.g., altered temperature-dependent activity profile, thermostability, pH activity, pH stability, substrate specificity, product specificity, and chemical stability.
There are, however, many limitations associated with expression screening using bacterial and yeast hosts; in some cases, the recombinant protein is in an inactive conformation, while in other cases the protein is unstable, or simply not synthesized in the first place. A protein of interest derived from a mammalian source is often poorly expressed in these hosts because either the protein expressed is in an inactive conformation or the protein is not expressed at all. In general, proteins derived from eukaryotes are expressed poorly in prokaryotic systems. Secondary modifications of recombinant proteins in these organisms may be very different from modifications that occur in the native host. Proteins from eukaryotes expressed in bacteria are unlikely to be correctly glycosylated, as these modifications occur in the secretory pathway, which is not present in bacteria. The most commonly used yeast strain, Saccharomyces cerevisiae, often hyper-glycosylates proteins, which can lead to expression of inactive proteins compared to their native states.
Another complication of using a bacterial or yeast host for enzyme screening is that the screening host is usually different from the final production host that is used industrially. There are important time and technical considerations involved in switching hosts. Even if an enzyme is successfully expressed in both types of hosts, expression constructs must be re-engineered when shifting from screening to production. More importantly, proteins that have been subjected to directed evolution, i.e., random mutagenesis, recombination, and selection of improved properties over generations of screening, may evolve to acquire improved properties that are specific to the screening host, perhaps by affecting folding and/or modification in a host-specific manner.
Another consideration for enzyme screening is the yield of expressed protein. While bacteria often produce very high yields of recombinant prokaryotic protein, poor yields of soluble, active protein are frequently observed when eukaryotic proteins are expressed in prokaryotes. Yeast hosts are often unable to support high levels of recombinant protein expression. Assays for biological activity during screening must be sufficiently sensitive to detect very low levels of protein. Often, detection is not possible, especially when the protein of interest has a poor specific activity. Furthermore, it is often advantageous to express proteins at a sufficient level such that detection of their activity can easily be distinguished over low levels of competing endogenous biological activity in the host strain. These concerns explain the frequent choice of a filamentous fungal host for production of eukaryotic genes, as these organisms often support high levels of protein expression. Screening is often crippled by poor yields obtained in commonly used yeast and bacteria hosts.
While filamentous fungal hosts have obvious advantages over bacterial and yeast hosts, high-throughput expression screening of filamentous fungal cultures is complicated by features of fungal morphology. For example, hyphae tend to clog pipet tips of liquid handlers, mycelial mats make it difficult to access liquid phase of cultures, and automated picking and inoculation of single filamentous fungal transformants on agar plates is not as routine as for yeasts and bacteria colonies.
Generating transformants that express a single type polypeptide is desirable when screening for novel or improved properties. Expression of a single type of polypeptide allows detection of small changes in protein performance. In contrast, working with transformed organisms that co-express more than a single type of polypeptide is technically challenging, because the screen sensitivity must be sufficient to detect a unique property of a protein that is present in a background of proteins having distinct properties. It is generally an advantage to have an expression screening host where a single type of polynucleotide fragment is introduced into each single transformant.
The process of introducing a single type of polynucleotide into a bacterial or yeast host is well known in the art. In bacteria, plasmid replicons can be used to prevent two different plasmids from coexisting in the same bacterial cell (Davidson, 1984 Gene 28: 1-15). Often, a library to be screened comprises a heterogenous population of plasmids, each including the same replicon, which is transformed into a pool of bacterial or yeast competent cells. When plasmids containing foreign genes are introduced into bacterial cells, the consequent outgrowth of the population leads to segregation of plasmids such that each cell contains only a single type of plasmid. Over the course of a few generations of bacterial growth, the minority plasmid is completely eliminated and the descendants of the original cell contain one plasmid or the other, but not both. Plasmids carrying the same replicon thus are said to belong to the same incompatibility group (Datta, 1979, in Plasmids of Medical, Environmental, and Commercial Importance, Timmis and Puhler, eds. Elsevier, Amsterdam).
Transformation of bacteria with a library of a heterogenous population of plasmids thus results in restriction of a single type of plasmid per transformant. On the other hand, the process of introducing a single type of polynucleotide into a filamentous fungal expression host is not as simple when transforming with a DNA library, which comprises a heterogeneous population of plasmids. When filamentous fungal strains are genetically engineered by introduction of foreign polynucleotide sequences, two different types of methods are routinely used. One method allows for integration of DNA into the fungal host chromosome. Frequently, more than a single polynucleotide fragment is introduced (Alesenko, 1994, Curr Genet 26:352-358). Another means of introducing the foreign DNA is to use an autonomously replicating plasmid. With both types of transformation, it is possible for a single host to contain more than one distinct polynucleotide fragment from the library that was used in transformation. The filamentous fungus is often multicellular and frequently multinucleate. It is common in the art to utilize spore purification in order to attempt to segregate unique polynucleotide fragments that were introduced into individual hosts. This process is time-consuming and difficult to automate, as colony picking robots do not easily pick filamentous fungal colonies for inoculation into liquid culture or for re-arraying for solid phase culture.
Restriction of genetic material so that individual transformants contain only a single type of polynucleotide fragment would be an advantage in the art of screening a filamentous fungal expression library. Specifically, it would be advantageous to have available a high-throughput method for transformation of filamentous fungi, where expression is relatively high, and where recovering genes from those transformants identified by a screen is fast and easy.
In addition to the advantages conferred in expressing individual DNA fragments from a DNA library in individual filamentous fungal transformants, it is also advantageous to have a facile method for recovering the foreign polynucleotide from transformants identified in the screen. Typically, a DNA rescue procedure is employed for plasmid transformants (such as AMA1 and ANS1 containing vectors) whereby a DNA preparation is made from the transformant, and this preparation is then used to transform E. coli in order to prepare suitable amounts of the plasmid for sequence analysis. For recovery of transformed DNA in filamentous fungal transformants where DNA has been integrated into the host chromosome, amplification of the foreign gene using PCR and genomic DNA prepared from the transformant is a commonly used method. Alternatively, one can prepare RNA from the transformant under conditions where the recombinant gene is expressed, and amplify the foreign polynucleotide from nucleic acid derived from the RNA. The common feature of all these nucleotide recovery methods is that they are time-consuming steps. It would be advantageous to have a quick and easy step for recovering the nucleotide material from an isolated filamentous fungal transformant that was derived from library expression screening.
WO 00/24883 discloses a general method for constructing and screening a cDNA library using an AMA plasmid in filamentous fungal cells.
An example of high throughput screening of filamentous fungal cultures is described by Lamsa and Bloebaum, 1990, J. Ind. Microbiol. 5: 229-238.
It is an object of the present invention to provide methods for single-well transformation and expression screening.